Melanoma refers to malignant neoplasms of melanocytes. Its proper diagnosis and early treatment by complete excision is of great importance because advanced melanoma has a poor prognosis and most melanomas are curable if excised in their early stages. In most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician. When the excision margins of a melanoma are identified based on this macroscopic appearance and no margin of seemingly uninvolved skin is excised, melanoma has the risk of local recurrence.
This has led to the recommendation to remove a safety margin of normal skin that varies from 0.5 to 3 cm depending on the thickness of the primary tumor (Wingo, P. A. et al., Cancer 82:1197-207 (1998); Rigel, D. S. et al., J Am Acad Dermatol 34:839-47 (1996); McGovern, V. J. et al., Cancer 32:1446-57 (1973)). It is obvious that the resulting defect inflicted by the excision can be considerable. If a melanoma measuring 2 cm in diameter that has a thickness of  greater than 4 mm is to be excised under the current guidelines, the resulting defect would be 8 cm (2+3+3 cm) in diameter. The closure of excisions with 2-3 cm margins usually require skin grafting and have the potential of adverse consequences such as unsatisfactory cosmetic result, increased morbidity and costs, and sometimes permanent functional impairment. Even with xe2x80x9cadequatexe2x80x9d safety margins, the melanoma can recur locally.
Obviously, it would be desirable if the margins could be tailored to the needs of the individual patient""s tumor. Unfortunately, so far, no technique exists that is able to detect the extent of a tumor accurately. In some types of melanomas the horizontally expanding portion of the tumor mainly consists of single melanocytes along the basal layer of the epidermis. These melanoma types are referred to as lentiginous melanomas. In these, the amount of atypical cells often gradually diminishes towards the margins so that it can be difficult or impossible for the pathologist to determine the border of the melanoma. However, current thinking implies that in most instances, the extent of a melanoma can be assessed by pathology. The fact that the removal of a margin of xe2x80x9chealthyxe2x80x9d skin reduces the recurrence rate, however, suggests that this skin is actually not healthy but contains residual melanoma which is undetectable by current methods.
The identification of useful means by which morphologically normal premalignant cells that have the capacity to form melanomas can be identified. The present invention addresses these and other needs.
The present invention provides methods of screening for the presence of premalignant melanocytes in a sample from a patient. The methods comprise contacting a nucleic acid sample from a biological sample from the patient with a probe which binds selectively to a target polynucleotide sequence on a chromosomal region which is amplified in melanoma cells. Usually, the copy number of the target sequence is determined. The nucleic acid sample is typically from morphologically normal cells adjacent to a melanoma lesion in the patient.
In the methods, the probe is contacted with the sample under conditions in which the probe binds selectively with the target polynucleotide sequence to form a stable hybridization complex and the formation of a hybridization complex is detected. The target sequence is selected from the group consisting of 11p15, 11q13, 22q12, 7p, 6p, 1q, 12q14, and 5p.
The nature of the nucleic acid sample is not critical to the invention. In some embodiments, the nucleic acid sample is a metaphase spread or an interphase nucleus. Typically, the probe is labeled e.g. with a fluorescent label. The label may be a direct label. Usually, a reference probe to a second chromosomal region (e.g. a centromere) is used in the methods as an internal control. In these embodiments, the second probe is labeled with a fluorescent label distinguishable from the label on the probe that selectively hybridizes to the target polynucleotide sequence.
In some embodiments, the probe may include repetitive sequences. In this case, the methods may further comprising the step of blocking the hybridization capacity of repetitive sequences the probe Unlabeled blocking nucleic acids comprising repetitive sequences (e.g. Cot-1 DNA) can be contacted with the sample for this purpose.
The nucleic acid hybridization can be carried out in a number of formats. For instance, the hybridization may be an in situ hybridization. In some embodiments, the probe is bound to a solid substrate e.g. in as a member of a nucleic acid array.
Definitions
To facilitate understanding the invention, a number of terms are defined below.
The term xe2x80x9campliconxe2x80x9d as used herein refers to a region of genomic nucleic acid which, when present in altered copy number, is associated with cancer. For example, the invention provides nucleic acid sequences which, when present in aberrant copy number, are associated with melanomas.
An xe2x80x9canimalxe2x80x9d refers to a member of the kingdom Animalia, characterized by multicellularity, the possession of a nervous system, voluntary movement, internal digestion, etc. An xe2x80x9canimalxe2x80x9d can be a human or other mammal. Preferred animals include humans, non-human primates, and other mammals. Thus, it will be recognized that the methods of this invention contemplate veterinary applications as well as medical applications directed to humans.
A xe2x80x9ccancerxe2x80x9d in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testis cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and the like.
The phrase xe2x80x9cdetecting a cancerxe2x80x9d refers to the ascertainment of the presence or absence of cancer in an animal, in this case, melanoma cells or premalignant melanocytes. xe2x80x9cDetecting a cancerxe2x80x9d can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal or to the likelihood or predilection to development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the animal.
The terms xe2x80x9chybridizing specifically toxe2x80x9d and xe2x80x9cspecific hybridizationxe2x80x9d and xe2x80x9cselectively hybridize to,xe2x80x9d as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. A xe2x80x9cstringent hybridizationxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I, chapt 2, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assays,xe2x80x9d Elsevier, N.Y. (xe2x80x9cTijssenxe2x80x9d). Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42xc2x0 C. using standard hybridization solutions (see, e.g., Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, e.g., Sambrook supra.) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4xc3x97 to 6xc3x97SSC at 40xc2x0 C. for 15 minutes.
The term xe2x80x9clabeled with a detectable compositionxe2x80x9d, as used herein, refers to a nucleic acid attached to a detectable composition, i.e., a label. The detection can be by, e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or chemical means. For example, useful labels include 32P, 35S, 3H, 14C, 125I, 131I; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense reagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. Dynabeads(trademark)), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label can be directly incorporated into the nucleic acid, peptide or other target compound to be detected, or it can be attached to a probe or antibody that hybridizes or binds to the target. A peptide can be made detectable by incorporating predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags). Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield (1995) Mol Cell Probes 9: 145-156.
The terms xe2x80x9cmelanomaxe2x80x9d or xe2x80x9ccutaneous melanomaxe2x80x9d refer to malignant neoplasms of melanocytes, which are pigment cells present normally in the epidermis and sometimes in the dermis. There are four types of cutaneous melanoma: lentigo maligna melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral lentiginous melanoma (AM). Melanoma usually starts as a proliferation of single melanocytes at the junction of the epidermis and the dermis. The cells first grow in a horizontal manner and settle an area of the skin that can vary from a few millimeters to several centimeters. As noted above, in most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician.
The term xe2x80x9cnucleic acidxe2x80x9d as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
The term a xe2x80x9cnucleic acid arrayxe2x80x9d as used herein is a plurality of target elements, each target element comprising one or more nucleic acid molecules (probes) immobilized on one or more solid surfaces to which sample nucleic acids can be hybridized. The nucleic acids of a target element can contain sequence(s) from specific genes or clones, e.g. from the regions identified here. Other target elements will contain, for instance, reference sequences. Target elements of various dimensions can be used in the arrays of the invention. Generally, smaller, target elements are preferred. Typically, a target element will be less than about 1 cm in diameter. Generally element sizes are from 1 xcexcm to about 3 mm, preferably between about 5 xcexcm and about 1 mm. The target elements of the arrays may be arranged on the solid surface at different densities. The target element densities will depend upon a number of factors, such as the nature of the label, the solid support, and the like. One of skill will recognize that each target element may comprise a mixture of nucleic acids of different lengths and sequences. Thus, for example, a target element may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths. The length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure, and to provide the required resolution among different genes or genomic locations. In various embodiments, target element sequences will have a complexity between about 1 kb and about 1 Mb, between about 10 kb to about 500 kb, between about 200 to about 500 kb, and from about 50 kb to about 150 kb.
The terms xe2x80x9cnucleic acid samplexe2x80x9d or xe2x80x9csample of human nucleic acidxe2x80x9d as used herein refers to a sample comprising human DNA or RNA in a form suitable for detection by hybridization or amplification. Typically, it will be prepared from a tissue sample from a patient who has or is suspected of having melanoma. The sample will most usually be prepared from tissue surrounding a melanoma tumor.
In many instances, the nucleic acid sample will be a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques. Alternatively, the nucleic acid may be isolated, cloned or amplified. It may be, e.g., genomic DNA, mRNA, or cDNA from a particular chromosome, or selected sequences (e.g. particular promoters, genes, amplification or restriction fragments, cDNA, etc.) within particular amplicons or deletions disclosed here.
The nucleic acid sample may be extracted from particular cells or tissues, e.g. melanocytes. Methods of isolating cell and tissue samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, needle biopsies, and the like. Frequently the sample will be a xe2x80x9cclinical samplexe2x80x9d which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes. The sample can also be derived from supernatants (of cells) or the cells themselves from cell cultures, cells from tissue culture and other media in which it may be desirable to detect chromosomal abnormalities or determine amplicon copy number. In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample may be isolated nucleic acids immobilized on a solid.
A xe2x80x9cpremalignant melanocytexe2x80x9d is a morphologically normal cell that has the capacity to form a malignant melanoma tumor. Such cells are typically found adjacent to a melanoma tumor. As used here, xe2x80x9cadjacentxe2x80x9d means less than 5 cm, usually less than 3 cm, from the nearest a typical cell in the tumor.
The term xe2x80x9cprobexe2x80x9d or a xe2x80x9cnucleic acid probexe2x80x9d, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected. The probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the regions described herein. The probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. The word xe2x80x9csamplexe2x80x9d may be used herein to refer not only to detected nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, e.g., with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately. What xe2x80x9cprobexe2x80x9d refers to specifically is clear from the context in which the word is used. The probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. In some embodiments, the probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854). One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are xe2x80x9csubstantially identicalxe2x80x9d to the disclosed probes, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived (see discussion above). Such modifications are specifically covered by reference to the individual probes described herein.
xe2x80x9cProviding a nucleic acid samplexe2x80x9d means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g. isolated by another person), or by performing the methods of the invention in vivo.
xe2x80x9cTissue biopsyxe2x80x9d refers to the removal of a biological sample for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.